This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have performed several pilot studies in Indian-origin rhesus macaques to determine safety and immunogenicity of the third-generation of the live attenuated, recombinant Listeria monocytogenes vector encoding SIV gag. This vaccine vector is called Lmdd-BdopSIVgag. First, we tested increasing doses of the vaccine vector to determine safety, immunogenicity, and optimal dose for oral vaccination. Each monkey received three vaccination courses on three sequential days at weeks 0, 6, and 19. We found that the new vaccine candidate was safe and immunogenic, and the higest dose was optimal (3 x 1012 colony forming units (CFU)). W then examined a different dose-schedule of the Lmdd-BdopSIVgag vaccine. Two groups of five animals were enrolled. The first group was immunized orally with the optimal dose (3 x1012 CFU) of Lmdd-BdopSIVgag. The second group received the "empty" vector, LmddBdop. Each monkey received four doses on alternative days at weeks 0 and 12. The analysis showed that this every-other-day schedule was not as good the administering the vaccine daily for three consecutive days. To test whether the animals in group 1 had been tolerized by the repeated exposure to the Listeria vaccine, we re-immunized these animals with Lmdd-BdopSIVgag according to the effiective daily x 3 schedule and enrolled a new group of 5 na[unreadable]ve monkeys that received the vaccine in parallel. A control group was given only empty vector. Good cellular immunity was seen in both vaccinated groups. These aniamls then received two mucosal boosts with live attuenated Adenovirus encoding SIV Gag (Ad5hrSIVGag). Strong increases in Gag-specific cellular immunity were seen. Our new data indicate that vaccination involving oral Listeria priming followed by mucosal adenovirus boosting induces strong cellular immunity. Our future plans will focus on inducing antibody responses by adding an immunization course with HIV envelope and Tat.